Proteolysis Targeting Chimeras (PROTACs) have brought fresh hope for treating diseases that were previously considered “undruggable.” They offer a promising alternative to traditional small-molecule inhibitors and biologics. But despite the excitement building around the potential of these therapies, preclinical evaluations of PROTACs can present distinct challenges, including pharmacokinetics (PK), pharmacodynamics (PD), safety, and bioanalytical hurdles.
If these challenges are not adequately addressed early in the development process, drug developers could face significant and costly delays in bringing PROTACs to market.
Why PROTACs are so promising
Before addressing challenges, it’s important to understand what makes PROTACs promising and how their unique mechanisms operate.
A PROTAC consists of two distinct components: one that binds to the target protein and another that recruits an E3 ubiquitin ligase. Together, these two molecules promote the ubiquitination of the target protein, marking it for degradation by the proteasome. PROTACs differ from traditional small-molecule inhibitors because they degrade proteins rather than merely block their function, offering a more permanent therapeutic effect.
This mode of action excites researchers who have found they can single out proteins that historically have been difficult to drug. These targeted proteins cause diseases such as cancer, neurodegenerative disorders, and autoimmune conditions.
However, their bifunctional structure, complexity, and reliance on intracellular machinery create unique hurdles that PROTAC developers must overcome if they are to fulfill their promise.
The key challenges in the preclinical development of PROTACs
PROTACs present several challenges to developers during preclinical development. Here are a few of the most significant.
Pharmacokinetics and biodistribution
Due to their large and complex structure, PROTACs suffer from issues with PK and biodistribution. Many PROTACs have poor bioavailability, limited cellular uptake, and rapid clearance from the body, which can hinder therapeutic efficacy. The unique bifunctional design of PROTACs also complicates absorption, distribution, metabolism, and excretion (ADME) profiles.
Cell permeability and target engagement
PROTACs’ cell permeability can negatively affect their ability to enter cells, bind to the target protein, and recruit the E3 ubiquitin ligase—particularly in tissues where penetration is difficult, such as the brain. The PROTACs’ size or polarity can prevent them from crossing plasma membranes. Once inside the cell, the extent and kinetics of target engagement vary depending on tissue-specific factors, making it challenging to predict therapeutic outcomes.
Off-target effects and toxicity
While PROTACs’ ability to degrade specific proteins is a major advantage, unintended degradation of off-target proteins can lead to significant toxicity. Off-target degradation could result from non-specific binding to other proteins or E3 ligases, leading to unintended ubiquitination. Preclinical toxicology studies are critical in identifying these off-target effects and ensuring that PROTACs are specific and safe. The potential for immunogenicity, especially in degrading proteins involved in immune regulation, must also be carefully evaluated.
Differences in E3 ligase expression between species
PROTAC efficacy relies on the presence of appropriate E3 ligases in target tissues. However, E3 ligase expression varies widely between species, which makes it more challenging to use animal models for preclinical evaluation. For example, ligases highly expressed in rodents may not be as prevalent in human tissues, or vice versa. This can complicate translating preclinical findings to human applications.
Drug-drug interactions (DDIs)
PROTACs can suffer from complex DDIs because they degrade target proteins, especially when co-administered with other therapies that rely on similar cellular pathways. For example, if the PROTAC degrades proteins involved in drug metabolism, this could affect the pharmacokinetics of other drugs, leading to altered efficacy or toxicity.
Bioanalytical challenges
For PK/TK bioanalysis, PROTAC recovery may be influenced by the linker’s instability and/or the E3 ligase cereblon (CRBN) ligand or non-specific binding with the experimental materials. Carryover may also occur during LC-MS/MS analysis due to the continuous binding and elution effects. Chiral bioanalysis should be performed if investigating in vivo chiral inversion of a PROTAC candidate is necessary. Developing chiral bioanalytical methods is a time-consuming “trial and error” process. The chromatographic peaks of the enantiomers should be baseline separated, which usually requires a relatively long elution time per sample. In contrast, most chiral stationary phase LC columns are not as stable and durable as ordinary LC columns.
Limited regulatory guidelines
Although PROTACs differ from traditional small molecules, they follow similar regulatory pathways. This means two species (one rodent and one non-rodent) are required for safety assessments for regulatory submission. Consequently, it’s critical to scientifically and strategically design the program, including selecting appropriate animal species, scientifically sound interpretation of toxicities obtained from nonclinical studies, and reasonable clinical application to humans.
Overcoming the challenges of PROTAC preclinical development
Challenges in the preclinical development of PROTACs can be overcome with scientific rigor, advanced analytical tools, and strategic partnerships with experienced and trusted advisors. Here are some key strategies.
Comprehensive PK/PD modeling
Advanced modeling techniques are required to handle PROTACs’ unique PK/PD profiles. In vitro and in vivo models should be used to predict how PROTACs will behave in different biological systems. By integrating these models with bioanalytical data, developers can gain valuable insight into PROTACs’ ADME properties, allowing for better optimization. PK/PD modeling can also inform dose selection and scheduling, ensuring targeted degradation is maximized and off-target effects are minimal.
Target engagement assays
Robust assays targeting engagement and protein degradation in relevant tissues are crucial to determining the efficacy of PROTACs. Western blotting, mass spectrometry, and proteomics are valuable tools for measuring protein levels in cells and tissues. They can help identify the concentration of PROTACs needed to achieve therapeutic effects while avoiding off-target activity.
Species-appropriate modeling
Preclinical studies should use models as close to human biology as possible. Humanized mouse models can provide more accurate predictions of PROTACs’ performance in human tissues. Researchers should also consider deploying in vitro models that use human cell lines, as they can also provide data on the efficacy and specificity of PROTAC-induced degradation.
Early DDI screening
Early screenings for potential interactions can mitigate the risk of DDIs. Researchers can use in vitro assays that assess PROTACs’ effects on key drug-metabolizing enzymes to identify possible interactions with other drugs. Early identification ensures risks are managed before clinical trials and reduces the chances of adverse reactions.
PK/TK bioanalysis
During biological sample treatment and analysis of PROTACs, the solvents, temperature, and experimental materials must be optimized to ensure that stability and non-specific binding issues do not compromise the accuracy and precision of the analysis.
Chiral bioanalytical methods
A rational strategy for employing chiral bioanalytical methods is to assess their necessity based on study objectives, using these methods only for chiral-related issues. The choice between validated and qualified methods should depend on research criticality to enhance development efficiency. Two advanced platforms are essential for chiral analysis of PROTACs in PK/TK bioanalysis. Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry(UPLC-MS/MS) is the gold standard for GLP-compliant studies, ensuring data integrity for regulatory submissions requiring stereochemical confirmation. Supercritical Fluid Chromatography-Tandem Mass Spectrometry (SFC-MS/MS), utilizing carbon dioxide(CO₂)-based mobile phases, offers faster chiral resolution while maintaining reproducibility, making it ideal for large-scale drug candidate screening and rapid lead optimization.
Partnering with an experienced testing partner
Partnering with a qualified and experienced preclinical testing laboratory is one of the most effective strategies for navigating the challenges of PROTAC development. A specialized service provider offers technical expertise, state-of-the-art technologies, and a deep understanding of the regulatory landscape. They can mitigate the challenges of PROTAC development using the solutions listed above, allowing developers to focus on the strategies required to bring the drug to market.
A final word
PROTACs’ promise lies in their ability to transform how we target and degrade disease-causing proteins, unlocking new possibilities for treating previously undruggable conditions. Yet, their complexity presents distinct preclinical challenges that demand innovative solutions. From optimizing PK/PD profiles and refining target engagement assays to mitigating species variability and drug-drug interactions, their success hinges on scientific precision and proactive strategy.
Drug developers can confidently navigate these challenges by leveraging cutting-edge bioanalytical tools, predictive modeling, and strategic partnerships with experienced preclinical testing organizations. With the right expertise and resources, the path from discovery to clinical success for PROTACs can be accelerated, turning potential into reality for the patients who need it most.
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